Acute pancreatitis is a severely debilitating, sometimes lethal disease in humans. Most therapies are supportive and target the hemodynamic effects such as dehydration with removal of precipitating factors that include, among others, alcohol or biliary obstructing calculi. Banks, P. A. 1997. Practice guidelines in acute pancreatitis. Am. J. Gastroenterol. 92:377-386; Baron, T. H., and Morgan, D. E. 1999. Acute necrotizing pancreatitis. N. Engl. J. Med. 340:1412-1417. There is therefore a long-felt unmet need for a novel therapy for the treatment of pancreatitis that provides more than palliative care. There is also a need for effective agents that can be used as prophylaxis towards pancreatitis in high risk patients.
Hemin up-regulates HO-1, a stress-induced enzyme implicated in protection from a variety of injuries while its related isoform HO-2 is constitutively expressed. Poss, K. D., and Tonegawa, S. 1997. Heme oxygenase 1 is required for mammalian iron reutilization. Proc. Natl. Acad. Sci. U.S.A. 94:10919-10924; Soares, M. P., Lin, Y., Anrather, J., Csizmadia, E., Takigami, K., Sato, K., Grey, S. T., Colvin, R. B., Choi, A. M., Poss, K. D., et al. 1998. Expression of heme oxygenase-1 can determine cardiac xenograft survival. Nat. Med. 4:1073-1077; Lee, T. S., and Chau, L. Y. 2002. Heme oxygenase-1 mediates the anti-inflammatory effect of interleukin-10 in mice. Nat. Med. 8:240-246; Otterbein, L. E., Soares, M. P., Yamashita, K., and Bach, F. H. 2003. Heme oxygenase-1:unleashing the protective properties of heme. Trends Immunol. 24:449-455.; Durante, W. 2003. Heme oxygenase-1 in growth control and its clinical application to vascular disease. J. Cell. Physiol. 195:373-382; Sikorski, E. M., Hock, T., Hill-Kapturczak, N., and Agarwal, A. 2004. The story so far: Molecular regulation of the heme oxygenase-1 gene in renal injury. Am. J. Physiol. Renal Physiol. 286:F425-441. Heme oxygenases catalyze the rate-limiting step in heme degradation to produce carbon monoxide (CO), iron, and biliverdin in equimolar amounts. Otterbein, L. E., et al., Trends Immunol. 24:449-455; Sikorski, E. M., et al., Am. J. Physiol. Renal Physiol. 286:F425-441; Tenhunen, R., Marver, H. S., and Schmid, R. 1968. The enzymatic conversion of heme to bilirubin by microsomal heme oxygenase. Proc. Natl. Acad. Sci. U.S.A. 61:748-755. Biliverdin is then reduced to bilirubin by biliverdin reductase. HO-1 (32 kDa; also referred to as heat shock protein-32) and HO-2 (36 kDa) are unique gene products that share nearly 40% identity at the amino acid level. Otterbein, L. E., et al., Trends Immunol. 24:449-455; Sikorski, E. M., et al., Am. J. Physiol. Renal Physiol. 286:F425-441. The role of HO-1 or hemin in the exocrine pancreas and their potential modulation of pancreatic injury are unknown. The only descriptions of HO-1 in the exocrine pancreas are two reports during 1997 that demonstrate HO-1 induction in caerulein-mediated mouse pancreatitis and in AR42J rat acinar cells exposed to H2O2 or CdCl2. Fu, K., Sarras, M. P., Jr., De Lisle, R. C., and Andrews, G. K. 1997. Expression of oxidative stress-responsive genes and cytokine genes during caerulein-induced acute pancreatitis. Am. J. Physiol. 273:G696-705; Sato, H., Siow, R. C., Bartlett, S., Taketani, S., Ishii, T., Bannai, S., and Mann, G. E. 1997. Expression of stress proteins heme oxygenase-1 and -2 in acute pancreatitis and pancreatic islet betaTC3 and acinar AR42J cells. FEBS Lett. 405:219-223, HO-1 null mice develop anemia and high tissue iron levels with consequent oxidative damage in the liver, glomerulonephritis and splenomegaly. These mice have >80% embryolethality and most surviving mice die within 6 months due to presumed multi-organ failure. Poss, K. D., and Tonegawa, S., Proc. Natl. Acad. Sci. U.S.A. 94:10919-10924; Soares, M. P., et al., Nat. Med. 4:1073-1077; Yet, S. F., Perrella, M. A., Layne, M. D., Hsieh, C. M., Maemura, K., Kobzik, L., Wiesel, P., Christou, H., Kourembanas, S., and Lee, M. E. 1999. Hypoxia induces severe right ventricular dilatation and infarction in heme oxygenase-1 null mice. J. Clin. Invest. 103:R23-29. There is also a single reported case of HO-1 deficiency identified in a child who died at the age of 6 with renal disease and intracranial hemorrhage. Yachie, A., Niida, Y., Wada, T., Igarashi, N., Kaneda, H., Toma, T., Ohta, K., Kasahara, Y., and Koizumi, S. 1999. Oxidative stress causes enhanced endothelial cell injury in human heme oxygenase-1 deficiency. J. Clin. Invest. 103:129-135; Kawashima, A., Oda, Y., Yachie, A., Koizumi, S., and Nakanishi, I. 2002. Heme oxygenase-1 deficiency: the first autopsy case. Hum. Pathol. 33:125-30. The effect HO-1 absence in humans or mice on pancreatic pathology is unknown.
Hemin is the prosthetic moiety for a broad range of proteins that play essential roles in oxygen delivery, mitochondrial function, and signal transduction including hemoglobin, cytochromes, prostaglandin endoperoxide and nitrous oxide synthases, catalase and peroxidases. Various formulations of hemin such as hematin and other alternate intravenous formulations (e.g. panhematin) are currently available and have been used in patients since the 1970's with reported minimal side effects to successfully treat acute porphyrias, to control liver allograft failure due to recurrence of erythropoietic protoporphyria, and in patients with thalassemia intermedia. Tenhunen, R., and Mustajoki, P. 1998. Acute porphyria: treatment with heme. Semin. LiverDis. 18:53-55; Dellon, E. S., Szczepiorkowski, Z. M., Dzik, W. H., Graeme-Cook, F., Ades, A., Bloomer, J. R., Cosimi, A. B., and Chung, R. T. 2002. Treatment of recurrent allograft dysfunction with intravenous hematin after liver transplantation for erythropoietic protoporphyria. Transplantation. 73:911-915; Rund, D., and Rachmilewitz, E. 2000. New trends in the treatment of beta-thalassemia. Crit. Rev. Oncol. Hematol. 33:105-118; Anderson, K. E., Bloomer, J. R., Bonkovsky, H. L., Kushner, J. P., Pierach, C. A., Pimstone, N. R., and Desnick, R. J. 2005. Recommendations for the diagnosis and treatment of the acute porphyrias. Ann. Intern. Med. 142:439-50. Hemin is also an established agent for HO-1 induction in several tested cultured cells and in vivo. Sato, H. et al., FEBS Lett. 405:219-223; Ishikawa, K., Sugawara, D., Wang, X., Suzuki, K., Itabe, H., Maruyama, Y., and Lusis, A. J.2001. Heme oxygenase-1 inhibits atherosclerotic lesion formation in ldl-receptor knockout mice. Circ. Res. 88:506-512; Kanakiriya, S. K., Croatt, A. J., Haggard, J. J., Ingelfinger, J. R., Tang, S. S., Alam, J., and Nath, K. A. 2003. Heme: a novel inducer of MCP-1 through HO-dependent and HO-independent mechanisms. Am. J. Physiol. Renal Physiol. 284:F546-554; Alam, J., Killeen, E., Gong, P., Naquin, R., Hu, B., Stewart, D., Ingelfinger, J. R., and Nath, K. A. 2003. Heme activates the heme oxygenase-1 gene in renal epithelial cells by stabilizing Nrf2. Am. J. Physiol. Renal Physiol. 284:F743-752; Graca-Souza, A. V., Arruda, M. A., de Freitas, M. S., Barja-Fidalgo, C., and Oliveira, P.L. 2002. Neutrophil activation by heme: implications for inflammatory processes. Blood. 99:4160-4165.
Conversion of trypsinogen to active trypsin within pancreatic acinar cells is an important event in developing acute pancreatitis. Furthermore, it has been well demonstrated that infiltrating neutrophils significantly contribute to this intrapancreatic trypsin activation. Gukovskaya, A. S., Vaquero, E., Zaninovic, V., Gorelick, F. S., Lusis, A. J., Brennan, M. L., Holland, S., and Pandol, S. J. 2002. Neutrophils and NADPH oxidase mediate intrapancreatic trypsin activation in murine experimental acute pancreatitis. Gastroenterology. 122:974-84. However, the mechanisms and signals that mediate neutrophil or other inflammatory cell (such as macrophage) recruitment into the pancreas are less well understood. Demonstrated herein is a novel protective role for hemin in experimental mouse pancreatitis and which provides a cellular mechanistic basis for such protection. This role is mediated by HO-1 and leads to recruitment of HO-1 expressing macrophages to the pancreas.